Method for forming an optimally exposed image of cytological specimen

ABSTRACT

First and second images of respective portions of a cytological specimen are acquired at different brightness levels. Sections of the respective first image are selected and combined with selected sections of the second image of each portion to form a composite image, so that non-nuclei cytological components that would otherwise appear dark in a single exposure image are brighter in the composite image.

FIELD OF INVENTION

The present invention relates to imaging and analysis of biologicalspecimens, and more particularly, to generating an optimally exposedimage of a specimen by combining sections of different images of thespecimen.

BACKGROUND

Medical professionals and cytotechnologists frequently review biologicalspecimens affixed to a specimen carrier, such as a slide, to analyzewhether a person from whom the specimen was obtained has or may have aparticular medical condition. For example, it is well known to examine acytological specimen in order to detect the presence of malignant orpre-malignant cells as part of a Papanicolaou (Pap) smear test. Tofacilitate this review process, automated systems have been employed toperform a pre-screening of the specimen slides in order to focus thecytotechnologist's attention on the most (or at least more) pertinentcells or groups of cells in the respective specimen, while discardingless relevant cells from further review. One such automated imagingsystem is the Thinprep Imaging System, available from Cytyc Corporation,250 Campus Drive, Marlborough, Mass. 01752 (www.cytyc.com).

More particularly, referring to FIG. 1, a cytological specimen 10 isprepared and carried by a slide 12. The cytological specimen 10 includesa number of portions 20, e.g., a first portion 21, a second portion 22,a third portion 23, etc. FIG. 1 generally illustrates square-shapedportions for purposes of illustration. The number of portions 20 canvary depending on the size of portions 20, the size of the specimen 10,and the size of the area of the specimen to be imaged. For example, aspecimen 10 can include about 3,000 portions 20 that are to be imaged.

Referring to FIG. 2, each portion 20 may include various cytologicalcomponents, including cells 30. Each cell 30 includes a nucleus 32 andcytoplasm 34 surrounding the nucleus 32. The area of a portion 20 thatis not occupied by cells 30 is generally referred to as background 36.The collection of portions that forms a specimen includes a collectionof cells 30 and background 36 areas.

Referring to FIG. 3, a specimen slide 12 is processed by an imagingsystem 40, such as the Thin Prep Imaging System. One exemplary imagingsystem 40 is also described in U.S. Pat. No. 7,006,674, the contents ofwhich are incorporated herein by reference as though set forth in full.An imaging system 40 typically includes a stage 42 upon which the slide12 is placed, a camera 44 for taking images of the specimen 10, and aprocessor or controller 46 for controlling the stage 42 and the camera46. The imaging system 40 can also include other components, asdescribed in the above-incorporated U.S. Pat. No. 7,006,674.

In use, the camera 44 acquires an image of a first portion 21 of thespecimen 10. The stage 42 is moved to move the specimen 10 so that thenext portion 22 is in view of the camera 44. An image of the second ornext portion 22 is acquired by the camera 44. The stage 42 is moved, andso on, until an image of each necessary portion 20 is acquired. In knownsystems, the image of each portion 20 is acquired at a particularbrightness or exposure level that is selected to be sufficiently brightso that most sections of the image (cytoplasm 34 and background 36) aresufficiently bright and visible.

For example, referring to FIG. 4, in one known imaging system, thecamera 44 is set to an exposure level of 229 based on a 0-255 grayscale.The result of this exposure setting is that the image of a portion 20 ofthe cytological specimen 10 has background 36 at a brightness level of229, cytoplasm 34 at a brightness level about 50 to 150, e.g., about 100to 150, and nuclei 32 at a brightness level of about 20. While thisexposure setting may be suitable for cytoplasm 34 and background 36,nuclei 32 in the image are not very visible since the brightness levelis only 20.

While known systems and methods have been used effectively to imagecells in the past, they can be improved. Initially, the resultingbrightness of different cytological components can vary significantly,and with known imaging systems, it is not always possible to seeindividual cell nuclei 32 and nuclei 32 that are clumped together. Forexample, as shown in FIG. 4, the brightness of the nuclei 32 in theimage is only about 20. Thus, the shape, size, color, and internalcomponents of nuclei 32 may not be observable. These shortcomings may bemore pronounced when attempting to view cell nuclei 32 that are clumpedtogether. Further, smaller nuclei 32 details may not be observable atall at these low brightness levels.

One potential solution to this problem is increasing the brightness orexposure so that the nuclei are brighter. However, this is not veryfeasible. If the exposure setting is 229, the exposure can only beincreased to 255 (i.e., about a 10% increase in exposure). This resultsin increasing the brightness of nuclei 32 by about 10%, i.e., increasingbrightness from 20 to about 22. This nominal increase in brightness isnot sufficient to provide the desired image quality and nuclei 32detail. Additionally, the camera cannot resolve anything brighter than255, and details in the brighter background and cytoplasm sections arelost as the pixel values peak at a brightness of 255. Thus, increasingthe increasing the exposure only provides minimal improvement to nuclei32 brightness while degrading other portions of the image.

It would be advantageous, therefore, to form an optimally exposed imageof a cytological specimen. It would also be advantageous to form animage with brighter nuclei and groups of nuclei without saturating otherparts of the image. It would also be desirable to illuminate nuclei andportions thereof that were not previously visible using known systems.

SUMMARY

According to one embodiment, a method for forming an optimally exposedimage of a cytological specimen includes acquiring a first and secondimages of a first portion of the specimen, wherein the first image isacquired at a first brightness level, and the second image is acquiredat a second brightness level higher than the first brightness level. Themethod further includes selecting and combining respective sections ofthe first and second images to form an optimally (or substantiallyoptimally) exposed image.

In one embodiment, the selected sections of the first image correspondto non-nuclei components of the first portion of the specimen, and theselected sections of the second image correspond to the nucleicomponents of the first portion of the specimen, wherein the selectedsections of the first and second images are combined to form anoptimally exposed composite image.

In one embodiment, the method further includes generating a mask of oneof the acquired images. The mask can be generated by passing one of theimages through a filter, such as a low-pass filter, and binarizing theoutput of the filter to generate the mask. The mask includes binaryvalues that identify nuclei and non-nuclei components of the firstportion of the image of the specimen. The method also includes selectingsections of the first image that correspond to non-nuclei components ofthe first portion of the specimen according to the mask and selectingsections of the second image corresponding to the nuclei components ofthe first portion of the specimen according to the mask. The selectedsections of the first and second images are combined to form anoptimally exposed image.

In various embodiments, the first and second images are images of cells.Nuclei of cells are more visible in the second image at the secondbrightness or exposure level than in the first image at the firstbrightness or exposure level, and non-nuclei components, such ascytoplasm and background, are more visible in the first image at thefirst, lower brightness level. For example, the first image at the firstbrightness level can include nuclei at a brightness of about 20,non-nuclei components at a brightness of about 50-150, and background ata brightness of about 229, based on gray scale range of 0-255, whereasthe second image at the second brightness level contains nuclei at abrightness of about 80, and non-nuclei components and background thatare saturated, based on a gray scale range of 0-255. The secondbrightness level can be brighter than the first brightness level byvarying degrees. In one embodiment, the second brightness level is aboutfour times as bright as the first brightness level. In anotherembodiment, the first brightness level is a non-saturation brightnesslevel, and the second brightness level is a saturation brightness level.

Sections of different images can be selected based on the types ofcomponents corresponding to particular sections. For example, sectionsof the first image at the first, lower brightness can correspond tonon-nuclei components of the specimen, and sections of the second imageat the second, higher brightness level can correspond to nucleicomponents of the specimen.

Sections of different images can be selected according to a mask, whichcan contain “0” and “1” values corresponding to different cytologicalcomponents. Certain mask values can represent nuclei components, andother mask values can represent non-nuclei components. The mask can begenerated based on either image. Further, the mask can be generated bybinarizing the output of a filter, such as a low pass filter, thatprocesses first and second image data. Using the mask, sections of thefirst image at the first brightness level corresponding to non-nucleicomponents are selected from the first image, and sections of the secondimage at the second brightness level corresponding to nuclei componentsare selected from the second image according to the mask. The selectedsections can then be combined to form an optimally exposed image.

Selected sections of different images can be combined in various ways.For example, selected sections of the first and second images can becombined by replacing certain pixels of one image with selected pixelsof another image, or merging selected pixels of the first and secondimages together. The steps of selecting sections of different imageportions and combining the selected sections can be repeated to processall of the first and second images.

Other aspects of embodiments are described herein and will becomeapparent upon reading the following detailed description with referenceto the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Referring now to the drawings in which like reference numbers representcorresponding parts throughout and in which:

FIG. 1 illustrates a slide having a cytological specimen and portionsthereof;

FIG. 2 illustrates components of a portion of a cytological specimen;

FIG. 3 generally illustrates a known imaging system for use ingenerating an image of a specimen at a single brightness level;

FIG. 4 illustrates brightness levels of components of a portion of animage of a cytological specimen in an image acquired at a singlebrightness level using a known imaging system;

FIG. 5 is a flow chart of a method of forming an optimally exposed imageof a cytological specimen according to one embodiment;

FIG. 6 illustrates application of light for different durations to allowimages of a portion of a cytological specimen to be taken at differentbrightness levels;

FIG. 7 illustrates application of light for different durations anddifferent amplitudes to allow images of a portion of a cytologicalspecimen to be taken at different brightness levels;

FIG. 8 shows one manner of forming a composite image;

FIG. 9 shows another manner of forming a composite image;

FIG. 10 generally illustrates a composite image of a cytologicalspecimen having sections of images taken at different brightness levelsaccording to one embodiment;

FIG. 11 is a flow chart showing a method of forming an optimally exposedimage of a cytological specimen according to another embodiment;

FIG. 12 generally illustrates an image of a portion of a cytologicalspecimen at a first brightness;

FIG. 13 generally illustrates an image of the portion of the cytologicalspecimen shown in FIG. 12 at a second brightness level that is higherthan the first brightness level;

FIG. 14 shows sections of a first image of a portion of a cytologicalspecimen corresponding to non-nuclei components;

FIG. 15 shows sections of a second image of the portion of thecytological specimen shown in FIG. 14 corresponding to nucleicomponents;

FIG. 16 shows a composite image that includes sections of the firstimage shown in FIG. 14 and sections of the second image shown in FIG. 15according to one embodiment;

FIG. 17 generally illustrates a system for generating a mask;

FIG. 18 is a flow chart showing a method of forming an optimally exposedimage of a cytological specimen according to another embodiment;

FIG. 19 illustrates generating a mask based on an image;

FIG. 20 illustrates a mask that is generated based on the image shown inFIG. 19;

FIG. 21 illustrates sections or pixels of the first image at a firstbrightness that are selected according to the mask shown in FIG. 20identifying non-nuclei components;

FIG. 22 illustrates sections or pixels of the second image at a secondbrightness that are selected according to the mask shown in FIG. 20identifying nuclei components;

FIG. 23 shows a composite image that includes sections of the firstimage shown in FIG. 21 and sections of the second image shown in FIG. 22according to one embodiment; and

FIG. 24 illustrates an exemplary imaging system in which embodiments ofthe invention can be implemented.

DETAILED DESCRIPTION OF ILLUSTRATED EMBODIMENTS

Embodiments of the invention improve upon known imaging systems byproviding methods for generating an optimally exposed image of acytological specimen, which includes selected sections of differentimages acquired at different brightness levels or exposures. Sections ofa first image at a first exposure level and sections of a second imageat a second brightness level are selected and combined to form acomposite image. Sections of the brighter image can be selected tobetter show darker components, such as nuclei, and sections of the otherimage can be selected to show components, such as cytoplasm andbackground, which are sufficiently bright without additional exposure.Such optimally exposed composite images include greater cytologicaldetail compared to images taken at a single exposure, which is achievedwithout saturating other parts of the image since sections in thebrighter image that might be saturated can be discarded, andnon-saturated sections of the image at the lower brightness level can beselected instead.

Referring to FIG. 5, one embodiment is directed to a method 500 forforming an optimally exposed image of a cytological specimen bycombining sections of different images at different brightness orexposure levels. In step 505, the camera and/or stage is positioned totake a first image of a first portion of cytological specimen, which canbe an image of an individual portion of the specimen, an image of agroup of portions of a specimen, and an image of all portions of thespecimen, i.e., an image of the entire specimen. For purposes ofexplanation, this specification refers to an image of an individualportion of a specimen. The first image is acquired at a first brightnessor exposure level (generally, “brightness level”). In step 510, afterthe first image of the first portion is acquired, the first brightnesslevel (B1) is adjusted to a second brightness level (B2), which isdifferent than B1. In step 515, a second image of the same first portionis acquired at B2. In step 520, sections of the first image of the firstportion are selected. In step 525, sections of the second image of thefirst portion are selected. In step 530, the sections selected from thefirst and second images of the first portion are combined into a firstcomposite image of the first portion of the specimen. In step 535, thestage is moved to position a second portion of the specimen for imaging.Steps 505-535, described with reference to the first portion, arerepeated for the second portion, repeated for the third portion, and soon for each additional portion of the cytological specimen that is to beimaged.

With the sequence shown in FIG. 5, the specimen is processedportion-by-portion, i.e., with sections of different images of a firstportion are selected and combined, followed by sections of differentimages of a second portion being selected and combined, and so forth foreach portion of the specimen. Alternatively, the steps shown in FIG. 5can be performed in different orders. For example, images of a pluralityof portions can be acquired, and after the images are acquired, they canthen be processed. Processing can involve selecting sections ofdifferent images of a first portion, selecting sections of differentimages of a second portion, and so on for other portions. The selectedsections can then be combined. Thus, for example, steps 520 and 525 canbe performed for each portion of the specimen, and then selectedsections of different images of the first portion, selected sections ofdifferent images of the second portion, and so on, can be combined atthe same time as step 530 to form an optimally exposed image. In otherwords, the combination of selected sections can occur after sections ofeach portion are selected or, alternatively, at the same time aftersections of all of the portions are selected. Accordingly, the methodshown in FIG. 5 applies to both portion-by-portion processing andprocessing that involves combining selected sections of multipleportions or all portions at the same or later time.

Referring to FIG. 6, one manner in which embodiments can be implementedis by changing the duration of an illumination pulse while the camera 44shutter is open, and the time during which the shutter is open can alsobe changed as necessary. As shown in FIG. 6, the camera 44 shutter isopen during times 602 and 604. During time 602, an image at a firstbrightness level or exposure can be acquired by an illumination pulse610 during time T1. During time 604, an image at a second brightnesslevel or exposure can be acquired using an illumination pulse 612 duringtime T2. In this example, the amplitudes of the pulses 612 and 614 arethe same and higher brightness levels are obtained by extending theduration of the illumination pulse. Persons skilled in the art willappreciate that different exposures can be generated by changing shutterspeeds and/or changing f/stop settings. Alternatively, the amplitude ofthe illumination pulse can be changed. Additionally, as shown in FIG. 7,both the amplitude and the duration of the illumination pulse can bechanged to provide different brightness levels.

According to one embodiment, B1 is the brightness level currentlyutilized by known systems, e.g., a level of 229, based on grayscale0-255. According to one embodiment, B2 is higher than B1. However, B2can be the brightness level currently utilized by known systems, and B1can be higher than B2. This specification refers to B2 as being brighterthan B1 for purposes of explanation, not limitation since “first” and“second” brightness levels are not intended to imply any particularorder or brightness hierarchy. The image acquired at the lowerbrightness level B1 can be taken first followed by the image at thehigher brightness level B2. Alternatively, the image at the higherbrightness level B2 can be taken first followed by the image at thelower brightness level B1. Accordingly, “first” and “second’ brightnesslevels and “B1” and “B2” as used in this specification refer todifferent brightness levels, not that the “first” level is brighter thanthe “second” level, and not that an image at a particular brightnesslevel must be taken before another image at another brightness level.Similarly, “first” and “second” images acquired at different brightnesslevels B1 and B2 as used in this specification refer to different imagesthat were acquired at different brightness levels.

Additionally, FIG. 5, step 530 (combining the selected sections of thefirst and second images of each image portion to form a composite image)can be performed in various ways. For example, referring to FIG. 8, theselected certain pixels of one image 800 can replace pixels of the otherimage 810. As shown in FIG. 8, selected sections or pixels (shown by“circled” B2 812) of the second image 810 acquired at B2 are replaced byselected sections or pixels 802 in the first image 800 that was acquiredat B1. Alternatively, selected sections or pixels 802 in the first image800 acquired at B1 can be replaced by selected sections or pixels 812 inthe second image 810 at acquired at B2. Additionally, referring to FIG.9, selected sections or pixels 802 in the first image 800 acquired at B1can also be merged or combined with selected sections or pixels 812 inthe second image 810 acquired at B2. Referring to FIG. 10, whether thereplacement (FIG. 8) or merging (FIG. 9) methods are utilized, acomposite image 1000 that includes selected sections or pixels 802 and812 of both images 800 and 810, shown by B1 and B2 pixel identifiers.

Referring to FIG. 11, in a method 1100 according to another embodiment,sections 802 and 812 of respective images 800 and 810 are selected onthe basis whether the sections 802 and 812 correspond to particularcytological components. In step 1105, a first image of a first portionof the cytological specimen is acquired at a first brightness (B1). Instep 1110, after the first image is acquired, the brightness isadjusted. For example, the exposure time and/or fstop can be changed tochange the brightness level from B1 and B2, e.g., as shown in FIG. 6. Instep 1115, a second image of the same first portion of the cytologicalspecimen is acquired at the second brightness (B2). For purposes ofexplanation, not limitation, this embodiment is described with referenceto the second brightness level is higher than the first brightnesslevel.

In step 1120, sections of the first image at the first brightness levelare selected if they correspond to certain cytological components.According to one embodiment, sections of the first image are selected ifthey correspond to non-nuclei components, such as cytoplasm andbackground. In step 1125, sections of the second image at the secondbrightness level (B2) are selected if they correspond to certaincytological components. According to one embodiment, sections of thesecond image are selected if they correspond to nuclei components. Thus,selected sections of the first image (at the lower brightness level)correspond to non-nuclei components, such as cytoplasm and background,and selected sections of the second image (at the higher brightnesslevel) correspond to nuclei. In step 1130, the selected portions of thefirst and second images are combined to form a composite image (e.g., asshown in FIGS. 8-10). The stage can be moved in step 1135 for imaging ofthe next specimen portion, and steps 1105-1135 can be repeated asnecessary to image additional portions of the specimen. Further, asdiscussed above with respect to FIG. 5, steps shown in FIG. 11 can beperformed in different orders.

FIGS. 12-16 further illustrate how different exposure levels areutilized to generate different images, select sections of the differentimages, and combine the selected sections to form a composite image.Referring to FIG. 12, according to one embodiment, the first image 800is acquired at a first brightness level B1 of 229, based on a grayscale0-255. Certain components of the first image 800 are brighter than othercomponents. In the illustrated embodiment, the brightest section is thebackground 36, which is “white” and has a brightness level of 229.Cytoplasm 34 is also relatively bright and has a brightness level ofabout 100. The nuclei 32 are the darkest components in the first image800 and have a brightness level of 20. Thus, the cytoplasm 34 is aboutfive times as bright as the nuclei 32, and the background 36 is about 11times as bright as the nuclei 20.

Referring to FIG. 13, the second image 810 is acquired at a secondbrightness level (B2). In this embodiment, B2 is higher than B1.According to one embodiment, B2 is sufficiently high to illuminatenuclei 20 so that nuclei can be viewed with the desired amount ofdetail. In one embodiment, B2 is a brightness that sufficientlyilluminates nuclei 32 while saturating other parts (such as cytoplasm 34and background 36) of the second image 810 since, according to oneembodiment, only the nuclei 32 are selected from the second image 810.Thus, cytoplasm 34 and background 36 being saturated in the second image810 does not affect the composite image 1000 since most, if not all, ofthe saturated sections of the second image 810 are not included in thecomposite image 1000.

In one embodiment, B2 is about four times as bright as B1. Inalternative embodiments, B1 can be increased by different amounts asnecessary to sufficiently illuminate nuclei 32 so that nuclei 32 can beviewed with the desired amount of detail. The resulting B2 values may ormay not saturate other cytological components depending on the dynamicrange of the camera 44. For example, in the embodiment shown in FIGS. 12and 13 involving a camera 44 having a dynamic range of 0-255 grayscale,B1 was increased by about 400%. Thus, relative to FIG. 12, thebrightness of the nuclei 32 in the second image 810 is increased by400%, or increased from 20 to 80. Increasing the brightness of thecytoplasm 34 and background 36 by 400% results in saturation of thesesections since, in this example, the dynamic range of the camera 44 is0-255. Consequently, increasing B1 by 400% would result in thebrightness of cytoplasm 34 and background 36 increasing well beyond thedynamic range (255) of the camera 44. Thus, in the second image 810, thecytoplasm 34 and background 36 are saturated at a brightness level of255, while the brightness of nuclei s about 80.

Referring to FIG. 14, in the illustrated embodiment, sections 802 of thefirst image 800 at the first brightness level (B1=229) corresponding tocytoplasm 34 at a brightness level of about 100 and background 36 at abrightness of about 229 are selected. Referring to FIG. 15, in theillustrated embodiment, only sections 812 of the second image 810 at thesecond brightness level (B2=4×B1) corresponding to nuclei 32 at abrightness level of about 80 are selected. As shown in FIG. 16, theseselected sections 802 and 812 of respective first and second images 800and 810 acquired at respective brightness levels B1 and B2 are combinedtogether to form a composite image 1000.

One manner in which a determination is made whether to select a section802 from the first image 800 acquired at B1 or to select a section 812from the second image 810 acquired at B2 is based on the cytologicalcomponent in each portion of the particular type of a cytologicalcomponent can be determined based on the brightness of a particularpixel or pixels occupied by the component. For example, a determinationcan be made whether the component is a nuclei 32 or a non-nucleicomponent (such as cytoplasm 34 and background 36) according to a mask.The mask can be generated based on one of the images and is a collectionof binary values (1s and 0s). The “1” values can identify nuclei 32 orportion thereof, and the “0” value can identify non-nuclei components.The mask values can be based on the brightness threshold in the imagethat is used to generate the mask.

For example, an imaging system can look at one of the images anddetermine which parts of the image have a brightness below a particularthreshold. Pixels having a brightness at or below the threshold aredetermined to be nuclei 32 components. These “nuclei” pixels are labeledwith a “1” value in the mask. Otherwise, pixels having a brightnessabove the threshold are determined to be non-nuclei components. These“non-nuclei” components are labeled with a “0” value I the mask. Thecollection of “0” and “1” pixel values form a mask, which can then beused to determine whether a given pixel of a composite image 1000 willbe populated by a corresponding pixel from the first image 800 or by acorresponding pixel from the second image 810. FIG. 17 illustrates anexemplary system for generating a mask.

Referring to FIG. 17, the camera 44 acquires an image 800 or 810 of thecytological specimen 10 on the slide 12. The image data (0-255brightness values) is passed through a filter, such as a low passspatial filter or converter or a median filter, to eliminate singlepixel “popcorn” type noise or single pixel noise elements. For example,if a pixel having a gray level brightness of 100 is inside a nucleusthat is predominately at a gray level brightness of 20, then the singlepixel at the gray level of 100 would not be considered to be a piece ofcytoplasm, and the filter 1700 would not permit that pixel to besegmented. The output of the filter 1700 is then binarized to create amask 1710.

According to one embodiment, the mask 1710 is based on the first image800 that is acquired at B1. In an alternative embodiment, the mask 1710is based on the second image 810 that is acquired at B2. The thresholdor filter cutoff values can be adjusted, as necessary, depending on thebrightness levels used in different images. Thus, FIG. 17 refers to theimage being the first image 800 or the second image 810 to indicate thateither image can be used to generate the mask 1710.

Referring to FIG. 18, according to one embodiment, a method 1800 offorming an optimally exposed image of a portion of cytological specimenincludes acquiring a first image of a first portion of the specimen atB1 in step 1805. In step 1810, a mask is generated based on the firstimage. As discussed above, the mask can be generated based on the secondimage at B2, but for purposes of explanation, this specification refersto generating the mask based on the first image at B1. In step 1815, thebrightness level is increased from B1 to B2. In step 1820, a secondimage of the same, first portion of the specimen is acquired at B2.

In step 1825, sections of the first image at B1 that correspond tonon-nuclei components of the first portion of the specimen are selectedaccording to the mask, i.e., based on the “0” values in the exemplarymask described above. In step 1830, sections of the second image at B2that correspond to nuclei components of the first portion of thespecimen are selected according to the mask, i.e., based on the “1”values in the mask. In step 1835, the selected sections of the first andsecond images are combined to form a composite image. The stage can bemoved in step 1140 for imaging of the next specimen portion, and steps1805-1840 can be repeated as necessary to image additional portions ofthe specimen. Further, as discussed above with respect to FIG. 5, stepsshown in FIG. 18 can be performed in different orders.

FIGS. 5, 11 and 18 show steps involving a second image at a secondbrightness level, but there may be times when an image at anotherbrightness level is not necessary. For example, this may occur is whenthe resulting mask includes all “0” values. Thus, rather than acquiringa second image at the second brightness level or exposure, the secondimage can be skipped and the system can proceed to the next location.

FIGS. 19-23 illustrate a further example of using a mask 1710 to selectsections 802 and 812 of first and second images 800 and 812 of portionsof the specimen 10 that correspond to nuclei 32 and non-nucleicomponents (e.g., 34 and 36). Referring to FIG. 19, one of the images800 or 810 is used to generate a mask 1710. For purposes of explanationand illustration, the image of a portion 20 of a specimen shown in FIG.19 is shown as having four nuclei 32 or groups of nuclei 32, which aresurrounded by cytoplasm 34 and background 36. As shown in FIG. 20, “lowbrightness” pixels are mapped to “1” values in the mask 1710 shown inFIG. 20. Similarly, the other pixels, which correspond to non-nuclei,such as cytoplasm 34 and background 36, are not passed through thefilter 1700 and are mapped to “0” values in the mask 1710 shown in FIG.20. Thus, the mask 1710 includes a collection of “0” and “1” valuesidentifying in the parent image (either the first image 800 or thesecond image 810) the locations of respective non-nuclei (34 and 36) andnuclei 32 components.

Having the mask 1710, referring to FIG. 21, sections 802 and 812 of eachimage 800 and 810 are selected according to the mask 1710. Thus, if themask 1710 shown in FIG. 20 were overlaid on the first image 800, pixelsor sections 802 (identified by B1) of the first image 800 at the first,lower brightness level are selected if a “0” value from the mask 1710 isassigned or mapped to that pixel or section 802. Similarly, as shown inFIG. 22, if the mask 1710 were overlaid on the second image 810, pixelsor sections 812 (identified by B2) of the second image 810 at thesecond, higher brightness level are selected if a “1” value from themask 1710 is assigned or mapped to that pixel or section 812. In theillustrated embodiment, selected pixels or sections 802 and 812 areexclusive so that when they are combined, a composite image 1000 isformed as shown in FIG. 23. Each pixel of the composite image 1000 ispopulated by a corresponding pixel or section 802 of the first image 800or a corresponding pixel or section 812 of the second image 810.

Persons skilled in the art will appreciate that only nuclei components32 can be mapped to “1” values and only non-nuclei components can bemapped to a “0” value. However, in some cases, it may be beneficial tomap areas surrounding a nucleus 32 to a “1’ nuclei value to make surethat all of the pixels of an image containing sections of nuclei 32,even small nuclei 32 sections, are selected and included in thecomposite image 1000. This also provides additional brighter sectionsaround the nucleus 32 to make it easier to ascertain nuclei 32boundaries. Accordingly, while various figures show selecting sections802 and 812 of first and second images 800 and 810 and mapping imagesbased on whether a particular pixel or section is a nuclei ornon-nuclei, persons skilled in the art will appreciate that the selectedor mapped “nuclei” sections 802 and 812 can extend to pixels containingonly small portions of nuclei 32 (e.g., a small portion of an outerboundary).

Further, persons skilled in the art will appreciate that embodiments ofthe invention can be applied to various imaging systems. One exemplaryimaging system 2400 is the system is shown in FIG. 24 and described inthe above-incorporated U.S. Pat. No. 7,006,674. Embodiments can beapplied to various other cytological imaging systems.

Although particular embodiments have been shown and described, it shouldbe understood that the above discussion is intended to illustrate andnot limit the scope of these embodiments, and various changes andmodifications may be made without departing from scope of embodiments.For example, embodiments can be applied to take different numbers ofimages of a specimen at different exposures. Further, the exposure ofeach image can be optimized to present the particular image componentsuch as nuclei, cytoplasm, white blood cells, different kinds of cells(e.g., squamous and endocervical cells that differ in brightness).

1. A method of generating a substantially optimally exposed image of acytological specimen, comprising: acquiring a first image of thespecimen at a first brightness level; acquiring a second image of thespecimen at a second brightness level higher than the first brightnesslevel; combining the respective sections of the first and second imagesto form a substantially optimally exposed image of the cytologicalspecimen; selecting sections of the first image which correspond tonon-nuclei components of the cytological specimen acquired at the firstbrightness level; and selecting sections of the second imagecorresponding to nuclei components of the cytological specimen acquiredat the second brightness level.
 2. The method of claim 1, wherein thefirst and second images are obtained of substantially a same portion ofthe specimen, the specimen portion containing both nuclei and non-nucleicomponents of individual cells.
 3. The method of claim 2, wherein nucleicomponents of the individual cells are more distinct in the second imagethan in the first image.
 4. The method of claim 2, wherein non-nucleicomponents of the individual cells are more distinct in the first imagethan in the second image.
 5. The method of claim 1, wherein the firstbrightness level is based on a first exposure time, and the secondbrightness level is based on a second exposure time different than thefirst exposure time.
 6. The method of claim 1, wherein the secondbrightness level is about four times greater than the first brightnesslevel.
 7. The method of claim 1, wherein the first brightness level is anon-saturation brightness level, and the second brightness level is asaturation brightness level.
 8. The method of claim 1, wherein therespective selected sections of the first and second images arecombining by replacing pixels of the selected sections of one of thefirst and second images with pixels of the selected sections of theother one of the first and second images.
 9. The method of claim 1,wherein the respective selected sections of the first and second imagesare combining by merging pixels of the selected sections of the firstimage with pixels of the selected sections of the second image.
 10. Themethod of claim 1, further comprising generating a mask that includespixels of the first image representing nuclei and non-nuclei componentsof the specimen.
 11. The method of claim 10, wherein the mask isgenerated by filtering and binarizing the first image, the maskincluding binary values representing both nuclei and non-nuclei cellscomponents.
 12. A method of generating a substantially optimally exposedimage of a cytological specimen, comprising: acquiring a first image ofthe specimen at a first brightness level; acquiring a second image ofthe specimen at a second brightness level higher than the firstbrightness level; and combining the respective sections of the first andsecond images to form a substantially optimally exposed image of thecytological specimen, wherein the first image containing cell nucleihaving a gray scale brightness value of approximately 20, non-nucleicell components having a grayscale brightness value in a range ofapproximately 50-150, and background having a gray scale brightness ofabout 229, respectively, based on a gray scale brightness range of0-255.
 13. The method of claim 12, the second image containing cellnuclei having a gray scale brightness value of approximately 80, andnon-nuclei cell components and background that are saturated,respectively, based on the gray scale brightness range of 0-255.
 14. Amethod of generating a substantially optimally exposed image of acytological specimen, comprising: acquiring a first image of a firstportion of the specimen, the first image being acquired at a firstbrightness level; acquiring a second image of the first portion of thecytological specimen, the second image being acquired at a secondbrightness level that is higher than the first brightness level;selecting sections of the first image corresponding to non-nucleicomponents of the first portion of the specimen; selecting sections ofthe second image corresponding to the nuclei components of the firstportion of the specimen; and combining the respective selected sectionsof the first and second images to form an substantially optimallyexposed image of the first portion of the specimen.
 15. The method ofclaim 14, wherein the second brightness level is about four times higherthan the first brightness level.
 16. The method of claim 14, wherein thefirst brightness level is a non-saturation brightness level, and thesecond brightness level is a saturation brightness level.
 17. The methodof claim 14, wherein nuclei components of the individual cells are moredistinct in the second image than in the first image, and whereinnon-nuclei components of the individual cells are more distinct in thefirst image than in the second image.
 18. The method of claim 14,wherein the respective selected sections of each of the respective firstand second images are combining by merging pixels of the selectedsections of the first image with pixels of the selected sections of thesecond image.